1. Field of the Invention
This invention relates to novel therapeutic compounds comprising a peptide, peptide analogue or organic chemical analogue targeted to megakaryocytes and platelets and a pharmaceutical agent, and to therapeutic compositions, polynucleotides encoding the compound and self-replicating polynucleotides capable of expressing the compounds. The therapeutic compounds are taken up and accumulate in the megakaryocytes that later produce platelets. The compounds of the invention are useful for inhibiting or countering vascular disorders such as thrombosis or atherosclerosis.
2. Description of the Background
Often the only effective means of treatment of the acute complications of cardiovascular diseases is the intravenous injection of a therapeutic agent. In many cases, however, these agents are somewhat toxic, and in some cases, highly toxic and their generalized distribution is not desirable. In addition, the intravenous injection of non-tissue targeted agents has other drawbacks such as the premature inactivation, catabolism and/or dilution of the agents, and the resulting lagtime in the delivery of the therapeutic agent. Higher doses, as well as a higher frequency of administration are thus required, because of its generalized distribution, to ensure the delivery of an adequate amount of drug to the target tissue. This further increases the exposure of non-target tissues to the drug and its toxic effects.
Megakaryocytes are bone marrow cells that mature into platelets. Megakaryocytes have .alpha.-granules that can accumulate some exogenous compounds such as fibrinogen. When megakaryocytes differentiate into platelets, the .alpha.-granules are dispersed into the maturing platelets. When deleterious events, such as injury or disease, occur within the circulatory system, they trigger the mobilization of platelets to the site of vascular damage. At the site of injury, the platelets are activated, e.g., to prevent blood loss through platelet aggregation and to release proteins active in coagulation. It is widely accepted nowadays that megakaryocytes do not synthesize the fibrinogen present in .alpha.-granules, but rather, acquire this protein by endocytosis. In addition to fibrinogen, the .alpha.-granules also contain, for example, platelet factor 4 (PF-4), and other proteins such as immunoglobulin G and albumin. The endocytosis of fibrinogen by megakaryocytes and platelets is thought to be mediated by the .alpha..sub.IIb .beta..sub.3 receptor, that is part of a family of cell surface receptors known as integrins. Platelets, and presumably megakaryocytes, also contain several other integrins, including receptors for vitronectin (.alpha..sub.v .beta..sub.3), collagen (.alpha..sub.2 .beta.1), fibronectin (.alpha.5.beta.1), and laminin (VLA-6). The vitronectin receptors, .alpha..sub.v .beta..sub.3, although present in low numbers, also bind fibrinogen.
Most cells synthesize their own proteins and store them in secretory granules. A few cell types such as basophilic leukocytes, mast cells, granule-containing lymphocytes, salivary gland cells, and megakaryocytes also incorporate exogenous proteins into their granules by endocytosis. However, little is known about the mechanisms and pathways involved in the uptake and incorporation of these exogenous proteins into the granules. The coated pit-mediated internalization of the LDL receptor was recently shown to require the presence of the consensus sequence NPXY, where X stands for any amino acid. The NPXY sequence was also found in cytoplasmic domains of some integrin subunits, including GP IIIa of platelets.
Fibrinogen constitutes ten percent of .alpha.-granule proteins and is the predominant adhesive protein secreted by platelets. Recent data indicate that fibrinogen is not synthesized by the bone marrow megakaryocytes, as previously thought, but is acquired exclusively from plasma by endocytosis. Because fibrinogen is present in the .alpha.-granules at a higher concentration than other proteins acquired exclusively from plasma, i.e., albumin, the endocytosis of fibrinogen is likely to be receptor-mediated. A variety of ligands, including fibrinogen, vWf, fibronectin, and thrombospondin, bind to the .alpha..sub.IIb .beta..sub.3 receptor on stimulated platelets. The recognition sequence common to these proteins is RGD. The fibrinogen molecule contains two RGD sequences both on the A.alpha. chain. Fibrinogen also contains an additional .alpha..sub.IIb .beta..sub.3 binding site located at the C-terminus of the .gamma. chain, which includes 12 amino acids but does not contain the RGD sequence. This dodecapeptide and the RGD-containing peptides compete with each other for binding to the .alpha..sub.IIb .beta..sub.3 receptor.
Attempts have been made to target drugs to specific tissues, such as cancer cells, for improved drug therapy (U.S. Pat. No. 5,135,736 to Anderson et al.). These cytotoxic agents designed to specifically bind target cells, however, may still need to be administered intravenously.
The administration of therapeutic agents for the treatment of cardiovascular diseases is often affected by the above described deficiencies, despite the intimate association of the circulatory system with the target tissue.
Accordingly, there is still a need for an improved therapeutic compound to more effectively treat cardiovascular diseases such as coronary thrombosis and atherosclerosis, which is specifically targeted for delivery to the diseased site and, therefore, produces minimal side effects.